Gα11-Phe220Ser Loss-of-Function Mutation Causes Familial Hypocalciuric Hypercalcemia Type-2 (FHH2) By Disrupting a Hydrophobic Cluster Critical for G-Protein Signaling

Presentation Number: SUN 245
Date of Presentation: April 2nd, 2017

Caroline M Gorvin*1, Treena Cranston2, Fadil Hannan3, Helena Valta4, Outi Makitie4, Camilla Schalin-Jantti5 and Rajesh V Thakker1
1University of Oxford, Oxford, United Kingdom, 2Oxford Molecular Genetics Laboratory, Churchill Hospital, Oxford, United Kingdom, 3University of Liverpool, Liverpool England, UNITED KINGDOM, 4University of Helsinki and Helsinki University Hospital, Helsinki, Finland, 5Helsinki University Hospital, Helsinki, Finland


Familial hypocalciuric hypercalcemia types 1-3 (FHH1-3) are due to loss-of-function mutations of the calcium-sensing receptor (CaSR), G-protein alpha-11 subunit (Gα11), and adaptor protein 2 sigma subunit (AP2σ), respectively. We investigated a kindred with FHH (4 affected and 2 unaffected members) which did not have CaSR or AP2σ mutations, for a Gα11 mutation, and identified a heterozygous Gα11 missense mutation, Phe220Ser, that is predicted to disrupt a cluster of hydrophobic residues within a domain important for G-protein coupled receptor binding. Gα11, which acts downstream of the calcium-sensing receptor (CaSR), activates: phospholipase C (PLC), to increase intracellular calcium (Ca2+i) release; and the extracellular-signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We assessed the effects of the Gα11 mutation on signalling via the PLC and ERK/MAPK pathways by expressing Gα11-wild-type Phe220 (WT), and three Gα11-mutants: Ser220, detected in the FHH2 kindred; and engineered mutants - Ala220 (a hydrophobic residue); and Glu220 (a non-hydrophobic residue), in HEK293 that stably expressed CaSR. Ca2+i responses to extracellular calcium (Ca2+e) were assessed using a Fluo-4 fluorescent assay and an NFAT-response element containing luciferase reporter that measures Ca2+i–induced gene expression; and MAPK responses were assessed using a phospho-ERK (pERK) AlphaScreen assay and a serum-response element (SRE) containing luciferase reporter that measures ERK-induced gene expression. Mutation of Phe220 to the non-hydrophobic Ser220 and Glu220 residues, but not the hydrophobic residue, Ala220, significantly impaired Gα11 signalling via PLC and ERK/MAPK pathways. Thus, Ser220 and Glu220, when compared to WT (Phe220) and Ala220 led to: a rightward shift of the dose-response curves of Ca2+i responses to Ca2+e with increased mean half-maximal concentration (EC50) values (Ser220 = 1.78mM (95% confidence interval (CI) =1.71-1.85)*, Glu220 = 1.71mM (95%CI=1.66-1.77)*, WT = 1.18mM (95%CI=1.13-1.24), and Ala220 = 1.14mM (95%CI=1.11-1.24), *p<0.001); reductions in NFAT reporter responses (maximal fold-changes (mean±SEM) to 5mM Ca2+e of Ser220 = 0.62±0.15*, Glu220 = 1.48±0.14$, WT = 2.08±0.13, and Ala220 = 1.96±0.11, *p<0.001, $p<0.05); impaired pERK responses (maximal fold-changes (mean±SEM) to 5mM Ca2+e of Ser220 = 4.12±0.71*, Glu220 = 2.41±0.65*, WT = 6.71±0.66, and Ala220 = 6.10±0.31, *p<0.001); and reductions in SRE reporter activity (maximal fold-changes (mean±SEM) to 5mM Ca2+e of Ser220 = 22.76±0.85*, Glu220 = 28.76±1.74$, WT = 35.04±1.71, and Ala220 = 36.74±5.44, *p<0.001, $p<0.05). Thus, we have identified a novel Gα11 mutation, Phe220Ser, causing FHH2, and have demonstrated the importance of the hydrophobic Phe220 residue, which forms part of a cluster, for G-protein signaling via the PLC and ERK/MAPK pathways.


Nothing to Disclose: CMG, TC, FH, HV, OM, CS, RVT